Methods and materials for treating cytokine release syndrome

ABSTRACT

This document relates to methods and materials involved in treating a mammal having, or at risk of developing, cytokine release syndrome (CRS). For example, a mammal having, or at risk of developing, CRS can be treated by administering one or more biguanides (e.g., metformin) to the mammal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/548,215, filed Aug. 22, 2019, which also claims the benefit of U.S.Provisional Application Ser. No. 62/724,930, filed Aug. 30, 2018. Thedisclosures of the prior applications are considered part of (and areincorporated by reference in) the disclosure of this application.

BACKGROUND 1. Technical Field

This document relates to methods and materials involved in treating amammal having, or at risk of developing, cytokine release syndrome(CRS). For example, a mammal having, or at risk of developing, CRS canbe treated by administering one or more biguanides (e.g., metformin) tothe mammal.

2. Background Information

CRS is a potentially life-threatening condition characterized by highfever and hypotension that may progress to multi-organ failure includingdistributive shock due to systemic capillary leak, renal failure andrespiratory failure that may lead to death (Porter et al., J HematolOncol. 2018;11(1):35). CRS is a potential complication of chimericantigen receptor T cell (CAR-T) therapy as well as the use of bispecificT-cell engagers (BiTE) such as blinatumumab (e.g., BLINCYTO®)(Shimabukuro-Vornhagen et al., J Immunother Cancer. 2018;6(1):56; andHoriuchi et al., J Biol Chem. 2017;292(20):8436-46). Perhaps 20% or moreof patients with hematologic malignancies treated with CAR-T cells endup in the intensive care unit for CRS (Park et al., N Engl J Med.2018;378(5):449-59).

SUMMARY

This document provides methods and materials for treating a mammalhaving, or at risk of developing, CRS. For example, a mammal having, orat risk of developing, CRS can be treated by administering one or morebiguanides (e.g., metformin) to the mammal. As demonstrated herein, IL-6receptor alpha (IL-6Rα; also known as CD126) expression on the surfaceof a cancer cell can indicate that the cancer cell is likely to respondto metformin. Also as demonstrated herein, metformin (a biguanideantihyperglycemic agent) can down-regulate IL-6R expression on thesurface of cells, and can prevent C-reactive protein (CRP) release fromthe liver in response to IL-6 stimulation of hepatocytes.

Patients are only treated for CRS once they meet certain criteria (e.g.,a certain severity of the CRS). In addition, patients having aninfection and CRS are typically excluded from CRS treatment since thesepatients are typically neutropenic and present with high fever. Havingthe ability to treat and/or prevent CRS as described herein provides anopportunity to administer immunotherapies (e.g., cancer immunotherapiessuch as BiTE technology and CAR-T cell therapy) with a reduced risk ofdeveloping CRS.

In general, one aspect of this document features methods for treating amammal having CRS. The methods can include, or consist essentially of,administering to a mammal in need thereof (e.g., a mammal having CRS) acomposition including a biguanide, where the severity of the CRS isreduced within the mammal. The mammal can be a human. The biguanide canbe metformin. The CRS can be associated with an immunotherapy. Theimmunotherapy can include CAR-T cell therapy. The immunotherapy caninclude BiTE therapy. The BiTE therapy can include administeringblinatumumab to the mammal.

In another aspect, this document features a method for treating a mammalat risk of developing CRS, where the mammal is scheduled to undergoimmunotherapy. The methods can include, or consist essentially of,administering to a mammal in need thereof (e.g., a mammal at risk ofdeveloping CRS) a composition comprising a biguanide. The mammal can bea human. The biguanide can be metformin. The CRS can be associated withan immunotherapy. The immunotherapy can include CAR-T cell therapy. Theimmunotherapy can include BiTE therapy. The BiTE therapy can includeadministering blinatumumab to the mammal. The composition including thebiguanide can be administered to the mammal prior to the immunotherapy.The composition including the biguanide can be administered to themammal concurrent with the immunotherapy. In some cases, the mammal doesnot develop said CRS following the administering step.

In another aspect, this document features a method for treating a mammalhaving cancer, where the cancer includes cancer cells having IL-6Rpolypeptides on the surface of the cancer cells. The methods caninclude, or consist essentially of, administering to a mammal in needthereof (e.g., a mammal having cancer), a composition including abiguanide. The mammal can be a human. The IL-6R polypeptides can beIL-6Rα polypeptides. The cancer can be a blood cancer. The blood cancercan be a multiple myeloma. The method also can include administering anagent used to reduce levels of IL-6R polypeptides to the mammal. Theagent can be an anti-IL-6R antibody. The anti-IL-6R antibody can betocilizumab. The mammal can be identified as having cancer cells havingIL-6R polypeptides on the surface of the cancer cells. The method caninclude identifying the mammal as having cancer cells having IL-6Rpolypeptides on the surface of the cancer cells.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 contains a graph showing a negative correlation between metforminIC₅₀ (as determined by MTT assay) and IL-6Rα (CD126) surface expression(as determined by flow cytometer). High cell surface expression ofIL-6Rα predicts for better sensitivity of the cell to metformin therapy(and at a lower dose).

FIG. 2 contains a graph showing a negative correlation betweenphenformin IC₅₀ (as determined by MTT assay) and IL-6Rα (CD126) surfaceexpression (as determined by flow cytometer).

FIGS. 3A-3B contain graphs comparing metformin efficacy in two primaryamyloidosis (AL) cell lines, ALMC1 and ALMC2, having different levels ofIL-6Rα (CD126). (FIG. 3A) Metformin IC₅₀ is lower for ALMC2 compared toALMC1 (as determined by MTT assay). (FIG. 3B) IL-6Rα expression ishigher for ALMC2 compared to ALMC1 (as determined by flow cytometer).

FIGS. 4A-4D show that metformin induced apoptosis and inhibition of cellcycle progression in myeloma cells independent of their underlyinggenetic abnormalities. (FIG. 4A) HMCLs were stained with IL-6Rα-PE andIL-6 signal transducer (IL-6ST)-APC antibodies and median fluorescenceintensity was determined for expression of cell surface markers usingflow cytometry. Data is presented as heat map intensity corresponding tothe level of expression. (FIG. 4B) HMCLs were treated with differentdoses of metformin for 72 hours and relative proliferation compared tovehicle control was calculated using MTT assay. Data is presented asIC₅₀ for different cell lines and IC₅₀ was calculated using Compusynsoftware (n=6). (FIG. 4C) & (FIG. 4D) HMCLs MM1S, RPMI, and U266 weretreated with vehicle control (DMSO) or metformin 10 mM for 72 hours.(FIG. 4C) For apoptosis assay, cells were suspended in Annexin V bufferand stained with Annexin V for 30 minutes. Propidium iodide was added 10minutes prior to data acquisition by flow cytometry. (FIG. 4D) For cellcycle assay, BrdU was added and incubated for 4 hours. Cells were fixed,permeabilized and stained with Anti-BrdU antibody for 30 minutes. 7AADwas added 10 minutes to samples before data acquisition by flowcytometry. The data were acquired on BD Fortessa and analyzed withflowjo V10.

FIGS. 5A-5F show that IL-6Rα expression on the myeloma cell surfacepredicted for response to metformin therapy and can serve as a biomarkerfor therapeutic response. Ten different HMCLs were treated withmetformin (0, 1 mM, 5 mM, 10 mM, 20 mM, 40 mM, and 80 mM) for 72 hours.The total numbers of live cells (metabolically active) were estimatedusing MTT assay. Relative proliferation was calculated by normalizingabsorbance value with that obtained for vehicle control. IC₅₀ wascalculated using Compusyn software. Basal cell surface expression ofIL-6Rα and IL-6ST was determined using flow cytometry. (FIG. 5A) Medianfluorescence intensity (MFI) for IL-6Rα was plotted against metforminIC₅₀ for different HMCLs (n=6 for IC₅₀ and n=3 for MFI) and thecorrelation determined (significant, negative correlation, p=0.0045).(FIG. 5B) MFI for IL-6ST was plotted against metformin IC₅₀ fordifferent HMCLs (n=6 for IC50 and n=3 for MFI) and correlationdetermined (not significant, p=0.7009). (FIG. 5C) IL-6Rα wasknocked-down (KD) using two different ShRNA against IL-6Rα. Decreasedexpression for IL-6Rα was confirmed with flow cytometry. (FIG. 5D) JJN3IL-6Rα over-expressing cells were created using IL-6R cDNA expressingvector. Increased expression of IL-6Rα was confirmed by flow cytometry.(FIG. 5E) The sensitivity to metformin (IC₅₀ ) was calculated using theMTT assay for cells transduced with the shRNA scrambled control orIL-6Rα KDs (n=6). Knockdown of IL-6Rα reduced the sensitivity (increasedthe IC₅₀) of RPMI cells to metformin compared to baseline. (FIG. 5F)Metformin IC₅₀ was calculated using MTT assay for JJN3 control andIL-6Rα over-expresser (n=6). Overexpression of IL-6Rα increased thesensitivity (decreased the IC₅₀) of the JJN3 cells to metformin.

FIGS. 6A-6E show that metformin killed myeloma cells by down-regulatingIL-6Rα expression and negating the growth and survival signal of IL-6 onmyeloma cells. RPMI, ANBL6, U266, MM1S, and JJN3 were treated withmetformin 10 mM for 72 hours and median fluorescence intensity for cellsurface expression of (FIG. 6A) IL-6Rα, (FIG. 6B) IL-6-Rβ and (FIG. 6C)CD44 was quantified using flow cytometry. Vehicle (open bar) andmetformin 10 mM (shaded bar), n=3. (FIG. 6D) RPMI and ANBL6 cells werecultured either in the presence of vehicle control or metformin 10 mMfor 72 hours. Soluble IL-6Rα was quantified in media supernatant usingELISA kit. Vehicle (open bar) and metformin 10 mM (shaded bar), n=6.Metformin decreased the concentration of soluble IL-6Rα in the media.(FIG. 6E) RPMI and ANBL6 cells were cultured either in presence ofvehicle control or metformin 10 mM for 72 hours. mRNA expression ofIL-6Rα was quantified using qPCR TaqMan assay. Vehicle (open bar) andmetformin 10 mM (shaded bar), n=6. Metformin decreased the expression ofIL-6Rα at the level of transcription.

FIGS. 7A-7B show that metformin killed primary myeloma cells bydown-regulating IL-6Rα expression. (FIG. 7A) Bone marrow aspirates wereenriched for multiple myeloma cells by with magnetic associated cellsorting using CD138⁺ (a marker for myeloma cells). Cells were treatedwith metformin 10 mM or vehicle control. After 48 hours of culture, theMTT assay was performed to determine the percentage of live cells. Datais presented as relative proliferation compared to vehicle control (n=6per sample). (FIG. 7B) CD138⁺ enriched cells were cultured for 48 hourseither with vehicle control or metformin 10 mM. Cells were stained withIL-6Rα-PE antibody. Data is presented as median fluorescence intensity(n=11). Metformin reduced cell surface expression of IL-6Rα on primarymultiple myeloma cells.

FIGS. 8A-8B show that metformin was effective in preventing C-reactiveprotein (CRP) release from hepatocytes after IL-6 stimulation. Hepatomacell line (Hep3b) was pre-treated with metformin (10 mM) or vehicle for72 hours. (FIG. 8A) Cells were stained with IL-6Rα-PE and data wasacquired on BD Fortessa. Data is presented as histogram. Unstainedcontrol was used as background control. Metformin decreased expressionof IL-6Rα on the surface of hepatocytes. (FIG. 8B) CRP release afterstimulation of hepatocytes with IL-6. After treatment equal numbers ofcells were seeded in each well (30,000 cells/ml) in 24-well plate. Cellswere treated for 48 hours with different combinations (Ctrl: Vehiclecontrol; Met: Metformin 10mM; IL-6: Interleukin 6 (50 ng/ml);Tocilizumab 10 μg/mL). Media were harvested and CRP was measured with ahuman CRP ELISA kit (Thermo Scientific) (n=4 per sample). Metforminpretreatment reduced CRP release from hepatocytes in response to IL-6stimulation. Metformin and tocilizumab reduced CRP production byhepatocytes to normal after stimulation with IL-6. In other words,metformin with tocilizumab neutralized the effect of IL-6 on hepatocyteCRP release.

DETAILED DESCRIPTION

This document provides methods and materials for treating a mammalhaving, or at risk of developing, CRS. For example, one or morebiguanides (e.g., metformin) can be administered to a mammal having CRSto treat the mammal. In some cases, one or more biguanides (e.g.,metformin) can be administered to a mammal at risk of developing CRS totreat the mammal.

Any appropriate mammal having, or at risk of developing, CRS can betreated as described herein. Examples of mammals having, or at risk ofdeveloping, CRS that can be treated as described herein (e.g., byadministering one or more biguanides such as metformin to the mammal)include, without limitation, humans, non-human primates (e.g., monkeys),dogs, cats, horses, cows, pigs, sheep, mice, and rats. For example, ahuman having, or at risk of developing, CRS can be treated byadministering metformin to that human.

When treating a mammal (e.g., a human) having, or at risk of developing,CRS as described herein (e.g., by administering one or more biguanidessuch as metformin to the mammal), the CRS can be any type of CRS. Incases where a mammal has CRS, the CRS can be any severity of CRS (e.g.,Grade 1, Grade 2, Grade 3, Grade 4, or Grade 5). In some cases, a CRScan be associated with administration of (e.g., can be a complicationof) an immunotherapy. Immunotherapy can be a cancer immunotherapy.Examples of immunotherapies include, without limitation, adoptive T celltherapies (e.g., CAR-T cell therapies such as tisagenlecleucel andaxicabtagene ciloleucel), antibody therapies (e.g., BiTE therapies suchas blinatumumab and solitomab), and immune effector cell therapies. Insome cases, CRS can be associated with an autoimmune disease and/or acomplication of an autoimmune disease (e.g., an arthritis such asrheumatoid arthritis and giant cell arteritis, and macrophage activationsyndrome). In some cases, CRS can be associated with transplantrejection (e.g., organ rejection, allograft rejection (e.g., allograftrejection after an allogeneic transplant such as a haploidenticaltransplant), host-versus-graft disease, and graft-versus-host disease(GVHD)). In some cases, CRS can be associated with an infection (e.g.,bacterial infections such as gram-positive bacterial infections andgram-negative bacterial infections, viral infections, protozoaninfections, and infections from higher organism such as helminthes).

In cases where a CRS is associated with an immunotherapy, and theimmunotherapy is a cancer immunotherapy, the cancer can be any type ofcancer. In some cases, a cancer can include one or more solid tumors. Insome cases, a cancer can be a blood cancer. Examples of cancers that canbe treated by a cancer immunotherapy include, without limitation,multiple myeloma (e.g., smoldering multiple myeloma), acutelymphoblastic leukemias (ALLs), chronic lymphocytic leukemias (CLLs),Hodgkin's lymphomas, non-Hodgkin's lymphomas, Waldenstrom'smacroglobulinemia, hairy cell leukemia, and primary amyloidosis (AL, AH,or AHL).

In cases where a CRS is associated with an immunotherapy, theimmunotherapy can target any appropriate antigen. Examples of antigensthat can be targeted by an immunotherapy include, without limitation,CD19, CD20, CD47, epithelial cell adhesion molecule (EpCAM), B cellmaturation antigen (BCMA), CD38, CD30, and epidermal growth factorreceptor (EGFR).

In some cases, methods described herein can include identifying a mammal(e.g., a human) as having CRS. Any appropriate method can be used toidentify a mammal having CRS. For example, detection of elevated levelsof biomarkers (e.g., cytokines such as IL-6, IFNγ, TNF-α, KC, MIP-2,and/or MIP-1β; ferritin; C-reactive protein; and fibrinogen) can be usedto identify a mammal having CRS. Any appropriate method can be used todetect elevated levels of biomarkers. Examples of methods that can beused to detect elevated levels of biomarkers include, withoutlimitation, western blotting techniques, enzyme-linked immunosorbentassay (ELISA), real-time polymerase chain reaction (PCR), flowcytometry, mass spectrometry, and a Luminex assay.

In some cases, methods described herein can include identifying a mammal(e.g., a human) as being at risk of developing CRS. For example, amammal undergoing, or scheduled to undergo, immunotherapy can be at riskof developing CRS. For example, a mammal undergoing, or scheduled toundergo, a transplant can be at risk of developing CRS. For example, amammal having high levels of proteins that are released by the liver(e.g., proteins that are released by the liver in response to elevatedlevels of circulating IL-6 levels such as CRP and ferritin) can be atrisk of developing CRS.

Once identified as having, or as being at risk for developing, CRS, amammal (e.g., a human) can be administered, or instructed toself-administer, one or more biguanides. For example, one or morebiguanides can be administered to a mammal in need thereof. A biguanidecan be any appropriate biguanide. In some cases, a biguanide can be ananti-hyperglycemic biguanide. In some cases, a biguanide can be aninhibitor of 5′ adenosine monophosphate activated protein kinase (AMPK)polypeptide expression. In some cases, a biguanide can be an inhibitorof AMPK polypeptide activity. Examples of biguanides that can be used asdescribed herein (e.g., to treat a mammal having, or at risk ofdeveloping, CRS) include, without limitation, metformin, phenformin,buformin, and derivatives thereof. For example, a mammal having, or atrisk for developing, CRS can be administered or can self-administermetformin.

When treating a mammal having, or at risk of developing, CRS, one ormore biguanides (e.g., metformin) can be administered to the mammal atany appropriate time. For example, when a mammal at risk of developingCRS is undergoing, or scheduled to undergo, immunotherapy, one or morebiguanides can be administered before, during (e.g., concurrent with),and/or after the immunotherapy. For example, when a mammal at risk ofdeveloping CRS is undergoing, or scheduled to undergo, a transplant, oneor more biguanides can be administered before, during (e.g., concurrentwith), and/or after the immunotherapy.

In some cases, treating a mammal having, or at risk for developing, CRSas described herein (e.g., by administering one or more biguanides suchas metformin to the mammal) can be effective to reduce the severity ofthe CRS and/or to reduce or eliminate one or more symptoms of the CRS.In some cases, the severity of CRS can be determined by the grade (e.g.,Grade 1, Grade 2, Grade 3, Grade 4, and Grade 5) of the CRS. In somecases, the severity of CRS can be as described elsewhere (see, e.g.,Porter et al, 2018, J Hematol Oncol. 11:35). Examples of symptoms of CRSinclude, without limitation, fever, fatigue, loss of appetite, muscleand joint pain, nausea, vomiting, diarrhea, rashes, fast breathing,rapid heartbeat, low blood pressure, seizures, headache, confusion,delirium, hallucinations, tremor, loss of coordination, low bloodoxygen, widened pulse pressure, altered cardiac output (e.g., increasedcardiac output in early CRS and decreased cardiac output in late CRS),high blood nitrogen levels, elevated levels of D-dimer polypeptides,elevated levels of transaminases, factor I deficiency, excessivebleeding, elevated levels of bilirubin, elevated levels of CRP, elevatedlevels of ferritin, elevated levels of IL-6, and elevated levels offibrinogen. In some cases, a symptom of CRS can be as describedelsewhere (see, e.g., Frey et al., 2016, Hematology Am Soc Hematol EducProgram. 2016:567-572).

In some cases, when treating a mammal having, or at risk for developing,CRS associated with a cancer immunotherapy as described herein (e.g., byadministering one or more biguanides such as metformin to the mammal),the treatment can reduce one or more symptoms of the cancer in themammal. For example, the treatment can reduce the number of cancer cellswithin a mammal. For example, the treatment can reduce the size (e.g.,volume) of one or more tumors within a mammal.

In some cases, when treating a mammal having, or at risk of developingCRS, as described herein (e.g., by administering one or more biguanidessuch as metformin to the mammal) can be effective to reduce the level ofIL-6R polypeptides (e.g., IL-6Rα) on one or more cells (e.g., on thesurface of one or more cells) within the mammal. For example,administering one or more biguanides to a mammal can reduce the level ofIL-6R polypeptide expression on any appropriate type of cell within themammal. Examples of cells on which one or more biguanides can reduce thelevel of IL-6R polypeptide expression include, without limitation,hepatocytes, epithelial cells, B-cells, plasma cells, monocytes, andmacrophages. Any appropriate method can be used to determine a level ofIL-6R polypeptide expression by cells within a mammal. Examples ofmethods that can be used to determine a level of IL-6R polypeptideexpression include, without limitation, western blotting techniques,ELISA, real-time PCR, flow cytometry, mass spectrometry, and a Luminexassay.

In some cases, treating a mammal having, or at risk of developing CRS,as described herein (e.g., by administering one or more biguanides suchas metformin to the mammal) can be effective to reduce the level of CRPpolypeptides (e.g., CRP polypeptides produced by one or more cells livercells) within the mammal. For example, administering one or morebiguanides to a mammal can reduce the level of CRP polypeptides withinthe mammal. Any appropriate method can be used to determine a level ofCRP polypeptides within a mammal. Examples of methods that can be usedto determine a level of CRP polypeptides include, without limitation,western blotting techniques, ELISA, real-time PCR, flow cytometry, massspectrometry, and a Luminex assay.

In some cases, one or more biguanides (e.g., metformin) can beformulated into a composition (e.g., a pharmaceutically acceptablecomposition) for administration to a mammal having CRS or being at riskfor developing CRS. For example, one or more biguanides can beformulated together with one or more pharmaceutically acceptablecarriers (additives) and/or diluents. Pharmaceutically acceptablecarriers, fillers, and vehicles that may be used in a pharmaceuticalcomposition described herein include, without limitation, dimethylsulfoxide (DMSO), ion exchangers, alumina, aluminum stearate, lecithin,serum proteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, andwool fat.

A composition including one or more biguanides (e.g., metformin) can bedesigned for oral or parenteral (including subcutaneous, intramuscular,intravenous, and intradermal) administration to a mammal (e.g., a human)having, or at risk of developing, CRS. Compositions suitable for oraladministration include, without limitation, liquids, tablets, capsules,pills, powders, gels, and granules. Compositions suitable for parenteraladministration include, without limitation, aqueous and non-aqueoussterile injection solutions that can contain anti-oxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient. In some cases, a composition includingone or more biguanides can be formulated for oral administration.

A composition including one or more biguanides (e.g., metformin) can bedesigned for any type of release (e.g., release of the one or morebiguanides from the composition) into the mammal the composition isadministered to (e.g., a mammal having, or at risk of developing, CRS).For example, a composition including one or more biguanides can bedesigned for immediate release, slow release, or extended release.

A composition including one or more biguanides (e.g., metformin) can beadministered locally or systemically to a mammal (e.g., a human) having,or at risk of developing, CRS. For example, a composition including oneor more biguanides can be administered locally to a mammal (e.g., ahuman) having, or at risk of developing, CRS by injection into or near abody part (e.g., tissue, cell type, or organ) of the mammal that isaffected by, or likely to be affected by, the CRS. For example, acomposition including one or more biguanides can be administeredsystemically by oral administration to a mammal having, or at risk ofdeveloping, CRS.

A composition including one or more biguanides (e.g., metformin) can beadministered to a mammal (e.g., a human) having, or at risk ofdeveloping, CRS in any appropriate dose(s). Effective doses can varydepending on the severity of the CRS, the risk of developing CRS, theroute of administration, the age and general health condition of thesubject, excipient usage, the possibility of co-usage with othertherapeutic treatments such as use of other agents, and the judgment ofthe treating physician. For example, in cases where a compositionincludes metformin, the composition can include from about 1 mM to about80 mM (e.g., from about 5 mM to about 80 mM, from about 10 mM to about80 mM, from about 15 mM to about 80 mM, from about 20 mM to about 80 mM,from about 30 mM to about 80 mM, from about 40 mM to about 80 mM, fromabout 50 mM to about 80 mM, from about 60 mM to about 80 mM, about 1 mMto about 70 mM, about 1 mM to about 60 mM, about 1 mM to about 50 mM,about 1 mM to about 40 mM, about 1 mM to about 30 mM, about 1 mM toabout 20 mM, about 1 mM to about 10 mM, about 5 mM to about 70 mM, about20 mM to about 60 mM, about 30 mM to about 50 mM, about 10 mM to about30 mM, about 20 mM to about 40 mM, about 40 mM to about 60 mM, about 50mM to about 70 mM, about 1 mM to about 80 mM, about 1 mM, about 5 mM,about 10 mM, about 20 mM, about 40 mM, or about 80 mM) metformin. Insome cases, a composition including metformin can include about 10 mMmetformin. For example, in cases where a composition includes metformin,the composition can include from about 500 mg to about 2500 mg (e.g.,from about 750 mg to about 2500 mg, from about 850 mg to about 2500 mg,from about 1000 mg to about 2500 mg, from about 1200 mg to about 2500mg, from about 1500 mg to about 2500 mg, from about 2000 mg to about2500 mg, from about 500 mg to about 2000 mg, from about 500 mg to about1800 mg, from about 500 mg to about 1500 mg, from about 500 mg to about1200 mg, from about 500 mg to about 1000 mg, from about 500 mg to about850 mg, from about 500 mg to about 750 mg, from about 600 mg to about2000 mg, from about 750 mg to about 1800 mg, from about 850 mg to about1500 mg, from about 1000 mg to about 1200 mg, about 500 mg, about 750mg, about 850 mg, or about 1000 mg) metformin. In some cases, acomposition including metformin can be a liquid including about 100 mgmetformin per mL liquid (e.g., about 500 mg metformin per 5 mL liquid).In some cases, a composition including metformin can be a tabletincluding about 500 mg metformin, 850 mg metformin, or 1000 mgmetformin. In some cases, a composition including metformin can be apill including about 500 mg metformin, about 750 mg metformin, or about1000 mg metformin. An effective dose of metformin can be a dailyeffective dose. In some cases, a composition including metformin can beadministered as single dose or a divided dose. An effective amount of acomposition including one or more biguanides can be any amount thatreduces the severity and/or one or more symptom of a condition beingtreated (e.g., CRS) without producing significant toxicity to themammal. The effective amount can remain constant or can be adjusted as asliding scale or variable dose depending on the mammal's response totreatment. Various factors can influence the actual effective amountused for a particular application. For example, the frequency ofadministration, duration of treatment, use of multiple treatment agents,route of administration, severity of the CRS, and risk of developing CRSmay require an increase or decrease in the actual effective amountadministered.

A composition including one or more biguanides (e.g., metformin) can beadministered to a mammal (e.g., a human) having, or at risk ofdeveloping, CRS in any appropriate frequency. The frequency ofadministration can be any frequency that reduces the severity of the CRSand/or one or more symptoms of the CRS without producing significanttoxicity to the mammal. For example, the frequency of administration canbe from about once a day to about ten times a day, from about threetimes a day to about eight times a day, or from about four times a dayto about six times a day. The frequency of administration can remainconstant or can be variable during the duration of treatment. As withthe effective amount, various factors can influence the actual frequencyof administration used for a particular application. For example, theeffective amount, duration of treatment, use of multiple treatmentagents, route of administration, severity of the CRS, and risk ofdeveloping CRS may require an increase or decrease in administrationfrequency.

A composition including one or more biguanides (e.g., metformin) can beadministered to a mammal (e.g., a human) having, or at risk ofdeveloping, CRS for any appropriate duration. An effective duration foradministering a composition including one or more biguanides can be anyduration that reduces the severity of the CRS and/or one or moresymptoms of the CRS without producing significant toxicity to themammal. For example, the effective duration can vary from several daysto several months or years to a lifetime. In some cases, the effectiveduration for the treatment of a CRS can range in duration from about 2days to about a week, from about 2 days to about a month, or from abouta week to about a month. Multiple factors can influence the actualeffective duration used for a particular treatment. For example, aneffective duration can vary with the frequency of administration,effective amount, use of multiple treatment agents, route ofadministration, severity of the CRS, and risk of developing CRS.

In some cases, one or more biguanides (e.g., metformin) can beadministered to a mammal having, or at risk for developing, CRS as thesole active ingredient. For example, one or more biguanides can beadministered to a mammal having, or at risk for developing, CRS as thesole active ingredient used to treat CRS. For example, one or morebiguanides can be administered as the sole active ingredient to a mammalin need thereof. For example, when one or more biguanides (e.g.,metformin) are administered to a mammal having, or at risk fordeveloping, CRS associated with a cancer immunotherapy, the one or morebiguanides can be the sole active ingredient used to treat cancer. Forexample, one or more biguanides (e.g., metformin) can be administered toa mammal having, or at risk for developing, CRS as the sole activeingredient used to reduce levels of IL-6R polypeptides (e.g., IL-6Rαpolypeptides).

In some cases, one or more biguanides (e.g., metformin) can beadministered to a mammal having, or at risk for developing, CRS with oneor more additional agents/therapies. For example, one or more biguanidescan be administered with one or more additional agents/therapies to amammal in need thereof. For example, one or more biguanides (e.g.,metformin) can be administered to a mammal having, or at risk fordeveloping, CRS with one or more agents that activate AMPK (e.g.,5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR)). For example, oneor more biguanides (e.g., metformin) can be administered to a mammalhaving, or at risk for developing, CRS with one or more agents thatinhibit mammalian target of rapamycin (mTOR; e.g., rapamycin). Forexample, one or more biguanides (e.g., metformin) can be administered toa mammal having, or at risk for developing, CRS with one or moreadditional agents/therapies used to treat CRS. Examples of additionalagents/therapies used to treat CRS include, without limitation,antibiotics (e.g., metronidazole, beta lactams such as penicillin,cephalosporins, carbapenems, fluoroquinolones, and macrolides),anti-histamines (e.g., chlorphenamine), corticosteroids (e.g.,hydrocortisone, prednisone, and dexamethasone), fever reducers (e.g.,acetaminophen), inotropic agents, hydration (e.g., intravenous fluidreplacement), and/or correcting overhydration (e.g., by dialysis or withfurosemide (e.g., intravenous furosemide)). For example, when one ormore biguanides (e.g., metformin) are administered to a mammal having,or at risk for developing, CRS associated with a cancer immunotherapy,the one or more biguanides can be administered with one or moreadditional agents/therapies used to treat cancer. Examples of additionalagents/therapies used to treat cancer include, without limitation,surgery, radiation therapies, chemotherapies, targeted therapies,hormonal therapies, angiogenesis inhibitors, immunosuppressants, immunecheckpoint inhibitors, immunomodulatory agents, and/or proteasomeinhibitors. For example, one or more biguanides (e.g., metformin) can beadministered to a mammal having, or at risk for developing, CRS with oneor more additional agents/therapies used to reduce levels of IL-6Rpolypeptides (e.g., IL-6Rα polypeptides). Examples of additionalagents/therapies used to reduce levels of IL-6R polypeptides include,without limitation, anti-IL-6 receptor antibodies (e.g., tocilizumab andsiltuximab). In cases where one or more biguanides (e.g., metformin) areused in combination with one or more additional agents/therapies, theone or more additional agents/therapies can be administered at the sametime or independently. For example, a composition including metformincan be administered first, and the one or more additionalagents/therapies can be administered second, or vice versa.

In certain instances, a course of treatment and the severity of one ormore symptoms related to the condition being treated (e.g., CRS) can bemonitored. Any appropriate method can be used to determine whether ornot the severity of a symptom is reduced. For example, the severity ofCRS can be assessed using any appropriate methods and/or techniques, andcan be assessed at different time points. For example, levels of CRP inthe blood (e.g., serum) of a mammal having, or at risk of developing,CRS can be assessed to determine the severity of the CRS.

In some cases, the methods and materials described herein can be used totreat a mammal having a disease or disorder associated with high levelsof IL-6 polypeptides. Examples of diseases and disorders associated withhigh levels of IL-6 polypeptides include, without limitation,Castleman's disease, neuromyelitis optica spectrum disorders (NMOSDs),refractory Behcet's uveitis, refractory major depressive disorder,schizophrenia, Erdheim-Chester disease, and monoclonal gammopathy ofundetermined significance (MGUS).

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 IL-6Rα (CD126) Expression and Metformin Response

Multiple myeloma is a heterogeneous disease with a complex molecularetiology with several groups characterized by recurrent chromosomaltranslocation. Consistent with preclinical and clinical studies in othertumor types, all myeloma cells do not have same sensitivity formetformin treatment. Most FDA approved drugs are not equally effectivein all patients; hence it is highly desirable to identify a marker thatcan predict metformin responders and achieve maximum benefits. Thisexample demonstrates that metformin is effective in inhibiting cellproliferation and increasing apoptosis in multiple myeloma. Thesensitivity of myeloma cells to metformin can be predicted by cellsurface expression of IL-6Rα that can serve as a biomarker for theefficacy of this drug.

Materials and Methods MTT Assay

(3-(4,5-Dimethylthiazol-2-5-Diphenyltetrazolium Bromide (MTT) (ThermoFisher Scientific, Mass., USA) was dissolved in PBS to make 12 mM stocksolution. Cells were seeded at 10,000 cells in 50 μl complete medium.Drugs, cytokines and antibodies were added at indicated doses in 50 μlcomplete medium. Cells were incubated for 72 hours for HMCLs and 48hours for primary myeloma cells in presence of vehicle control, ormetformin (1 mM, 5 mM, 10 mM, 20 mM, 40 mM, and 80 mM). MTT (10 μl) wasadded to each well and incubated for an additional 2 hours. Lysis buffer(100 μl) was added to each well and absorbance was read at 570 nm.

Flow Cytometry

Cell surface expression of IL-6Rα, IL-65T, and CD44 was quantified usingthe fluorescently labeled antibodies anti-IL6R-PE (UV4, Biolegend),anti-IL6ST-APC (2E1B02, Biolegend) and anti-CD44-BV711 Biolegend), andcompared with isotype control. BrdU labeling assay (Phase-Flow™,Biolegend) was performed according to the manufacturer's protocol toquantify cell cycle. After drug treatment, cells were labeled with BrdUfor 2 hours. For apoptosis assay, the cells were treated with metforminfor 72 hours and quantified with Annexin V (Biolegend). All flowcytometry experiments were on an LSR Fortessa (Becton Dickinson). Thedata were analyzed using flowJo vs10.4.2 (Flowjo LLC, USA).

Results

IL-6Rα, also known as CD126, is expressed primarily on hepatocytes,epithelial cells, plasma cells, and B-cells.

Cell surface expression for IL-6Rα was measured for 10 different humanmyeloma cell lines (HMCLs) by flow cytometer. IL-6Rα expression variedacross HMCLs (FIG. 4A). HMCLs were treated with different doses ofmetformin for 72 hours and MTT assay was conducted. IC₅₀ was calculatedusing Compusyn software. Metformin IC₅₀ showed strong negativecorrelation to IL-6Rα expression (FIG. 1). RPMI and KMS12PE with highestIL-6Rα expression required less than 10 mM metformin to achieve 50% celldeath/growth inhibition. JJN3 with no significant IL-6Rα expressionrequired more than 30 mM metformin for 50% cell death/growth inhibition.

Phenformin, a biguanide having similar mechanism to metformin but havingmore potency, was also examined. Phenformin IC₅₀ also stronglynegatively correlated with IL-6Rα expression (FIG. 2). IL-6Rα highexpressers were also better phenformin responders.

Primary amyloidosis (AL) is plasma cell disorder. Two AL cell lines,ALMC1 and ALMC2, from a patient were characterized. ALMC1 and ALMC2expressed significantly different level of IL-6Rα (FIG. 3B). ALMC2, ahigh IL-6Rα expresser, responded better to metformin than low IL-6Rαexpresser ALMC1 (FIG. 3A).

These results demonstrate that IL-6Rα cell surface expression level canpredict metformin response in multiple myeloma and primary amyloidosis.Further, metformin was effective in inhibiting cell proliferation andincreasing apoptosis in multiple myeloma.

Example 2 Diagnostic and Therapeutic Implications of Targeting IL-6 withMetformin Materials and methods Cell Culture

Human myeloma cells lines (HMCLs) RPMI-8266, U266-B1 and MM1S werepurchased from American Type Culture Collection (ATCC, Manassas, Va.,USA). OPMI, OPM2, ANBL6, OCI-MYS, JJN3 and KMS12PE were obtained fromdifferent laboratories at Mayo Clinic. HMCLs were maintained inRPMI-1640 (Thermo Scientific, Grand Island, N.Y., USA) mediumsupplemented with 10% heat-inactivated fetal bovine serum (FBS) (ThermoScientific), 100 U/mL penicillin, and 100 μg/ml streptomycin (ThermoScientific) in a humidified incubator and 5% CO₂ at 37° C. ANBL6 cellswere also supplemented with 1 ng/ml recombinant human IL-6 (Biolegend,San Diego, Calif., USA).

Human hepatocellular carcinoma cell line Hep3B was purchased from ATCC.Cells were maintained in DMEM supplemented with 10% FBS and 100 U/mLpenicillin and 100 μg/mL streptomycin.

Primary Multiple Myeloma cells

Approval was obtained from Institution Review Board and all patientsprovided informed consent. CD138⁺ cells from bone marrow aspirates wereenriched using magnetic associated cell sorting at the HematologyResearch Core (Mayo Clinic Rochester, USA). Primary myeloma cells weremaintained in RPMI supplemented with 10% FBS, antibiotics and 1 ng/mLIL-6.

Inhibitors and Drugs

Metformin (Millipore, Darmstadt, Germany) was dissolved in DMSO. Actemra(tocilizumab) was obtained from the Mayo Clinic Pharmacy.

MTT Assay

(3-(4,5-Dimethylthiazol-2-5-Diphenyltetrazolium Bromide (MTT)(ThermoFisher Scientific, Mass., USA) was dissolved in PBS to make 12 mMstock solution. Cells were seeded at 10,000 cells in 50 μL completemedium. Drugs, cytokines and antibodies were added at indicated doses in50 μL complete medium. HMCLs were incubated for 72 hours while primarymyeloma cells were incubated for 48 hours in presence of vehiclecontrol, metformin (1 mM, 5 mM, 10 mM, 20 mM, 40 mM and 80 mM). MTT (10μL) was added to each well and incubated for an additional 2 hours.Lysis buffer (100 μL) was added to each well and absorbance was read at570 nm. Absorbance under treatment was normalized with average ofvehicle control and presented as relative proliferation with respect tocontrol. IC50 was calculated using Compusyn software.

Flow Cytometry

Surface expression of IL-6Rα, IL-6ST and CD44 was quantified usingIL-6Rα-PE (clone UV4, Biolegend), IL-6l ST-APC (2E1B02, Biolegend) andCD44-BV711 (IM7, Biolegend), and compared to isotype or unstainedcontrol. BrdU labelling assay (Phase-Flow™, Biolegend) was performedaccording to the manufacturer's protocol to quantify cell cyclefractions. After treatment cells were labelled with BrdU for 2 hours.For apoptosis assay, cells were treated with metformin for 72 hours andquantified with Annexin V (Biolegend). All assays were quantified on LSRFortessa. Data were analyzed using flowJo vs10.4.2 (Flowjo LLC, USA).

Enzyme-Linked Immunosorbent Assay

A human soluble IL-6Rα ELISA kit was purchased from ThermoFisherScientific. Cells were cultured with or without treatment in 12-wellplates for 48 hours. Cell conditioned media were collected and proteinlysates was prepared from cell pellets. Standard and reagents wereprepared as per manufacturer's instructions. Undiluted 100 sample orstandard was added per well in duplicates and 40 μL of HRP conjugate toeach well. The plate was incubated for 2 hours on a mechanical shaker.After multiple washing, 100 μL TMB substrate solution was added to eachwell and incubated for 10 minutes in the dark. 100 μL of stop solutionwas added to each well and absorbance was read at 450 nm. Absoluteconcentrations of sIL-6R were interpolated from the standard curve.

A CRP ELISA kit was purchased from ThermoFisher Scientific.Hepatocellular cells (Hep3B) were treated either with metformin 10 mM orvehicle control for 72 hours. After pre-treatment, cells were countedand seeded in 24-well plates at density of 30,000 cells in 1 mL culturemedium. Metformin pre-treated cells were used for metformin (10 mM),IL-6 (50 ng/ml)+Metformin (10 mM) and IL-6 (50 ng/ml)+Metformin (10mM)+Tocilizumab (10 μg/ml) group. Vehicle pre-treated cells were usedfor control, IL-6 (50 ng/ml), IL-6 (50 ng/ml)+Tocilizumab (10 μg/ml)group. After 48 hours of culture, medium was harvested from each groupand CRP level was quantified using ELISA kit as per manufacturer'sinstruction.

Gene Expression

TaqMan assay and relative method (ddCT) were used to quantify geneexpression. Total RNA was extracted using RNeasy (Qiagen) kit andquantified for amount and purity with Nanodrop. 2 μg of RNA wasconverted to cDNA using first strand High-Capacity cDNA reversetranscription kit (ThermoFisher). 10 ng/well in 10 μL reaction per wellin duplicate for each samples were used to quantify mRNA. TaqMan FastAdvanced Mastermix (Applied Biosystems) was used and plates were run onViiA 7 Real-Time PCR system (ThermoFisher Scientific). Data wasnormalized with two independent house-keeping genes (HPRT1 and ACTB).

IL-6Rα Knock-Down (KD)

pGFP-C-shLenti vectors (4 shRNA targeting IL-6Rα and 1 scramble control)were purchased from Origene. Lentiviral particles were generated inHEK293T cells using pMD2.G (Addgene) and psPAX2 (Addgene). HMCLs weretransduced with lentiviral vector particles in the presence of Polybrene(Millipore). Stable cell lines were generated after selecting for GFPpositive cells with flow sorting and treatment with puromycin. Decreasedexpression of IL-6Rα compared to scrambled control was confirmed withqPCR and flow cytometer.

IL-6Rα Over-Expression

pCMV3-IL-6R-OFPSpark was purchased from Sinobiological (Wayne, Pa.,USA). JJN3 cells were transfected with pCMV-IL-6R plasmid using HDfugene. After geneticin selection, cells were sorted for RFP positivepopulation. IL-6Rα over-expression was confirmed with flow cytometry.

Data Analysis

All experiments were replicated multiple times. GraphPad Prism (v-7.03)was used to generate graph. Data is presented as mean±SEM (standarderror of the mean). Student “t” test was used to calculate statisticalsignificance between two groups and ANOVA to compare more than twogroups. ***P<0.001, **P<0.01 and *P<0.05.

Results

Metformin Induced Apoptosis and Inhibited Cell Cycle Progression inMyeloma Cells Independent of their Underlying Genetic Abnormalities.

Toxicity associated with metformin was evaluated in hematologicmalignancies, specifically multiple myeloma as well as other plasma celldisorders and cell based therapy for cancer such as chimeric antigenreceptor T cells (CAR-T). The in vitro activity of metformin was studiedagainst 10 different human multiple myeloma cell lines (HMCLs) thatrepresent the full spectrum of cytogenetic and FISH abnormalities thatoccur in multiple myeloma: from hyperdiploidy to translocations such ast(11;14); t(4;14); t(14;16) and deletion of the short arm of chromosome17 (17p) that is the locus of TP53, perhaps the most important tumorsuppressor gene known to humans (Table 1).

TABLE 1 Characterization of HMCLs used in these studies. Chromosomalabnormality (CA), targeted genes, RAS and p53 mutations status (wildtype (WT) and mutated (Mut)). All three groups of HMCLs based on IL-6signaling (independent, autocrine and dependent) were included. HMCLs CATarget genes RAS TP53 IL6 group KMS12PE t(11:14) CCND1 WT MutIndependent RPMI t(14:16) c-Maf Mut Mut Independent U266 t(11:14) CCND2WT Mut Autocrine ANBL6 t(14:16) c-Maf WT Mut Dependent MM1S t(14:16)c-Maf Mut WT Independent OPM1 t(4:14)  MMSET/FGFR3 WT Mut IndependentOPM2 t(4:14)  MMSET/FGFR3 WT Mut Independent KMS11 t(4:14)  MMSET/FGFR3WT Mut Independent OCI-MYS t(14:16) c-Maf WT Mut Independent JJN3t(14:16) c-Maf Mut Mut Independent KP6 Hyperdiploid

In vitro studies showed that metformin inhibited survival/growth ofmultiple myeloma cells with variable levels of efficacy (FIG. 4B). Theeffect of metformin on two hallmarks of cancer cells, cell proliferationand apoptosis, was also analyzed. In MM1S, RPMI and U266 cells,metformin increased apoptosis (FIG. 4C). The percentage of live cellsafter metformin treatment decreased to 59% in RPMI while in U266 andMM1S decreased to 67% and 76% respectively. Next the effect of metforminon cell cycle progression was examined using the BrdU assay. BrdU assayis also used clinically in myeloma at diagnosis and at relapse todetermine the ‘S phase’ fraction. Similar to what was observed in theapoptosis assay, a reduction in the S phase fraction was found inresponse to metformin from 53% to 29% in RPMI, 35% to 20% in U266 and34% to 23% in MM1S after metformin treatment (FIG. 4D) (all changes arehighly statistically significant).

IL-6Rα Expression on the Myeloma Cell Surface Predicted for Response toMetformin Therapy and Can Serve as a Biomarker for Therapeutic Response.

HMCLs were evaluated for basal expression of several components of IL-6signaling (IL-6, IL-6Rα, and IL-6ST) (FIG. 4A) and CD44. HMCLs thatrepresent the full spectrum of multiple myeloma subtypes and havefrequent genetic abnormalities including mutations in TP53 and KRAS wereincluded (Table 1). Irrespective of the underlying chromosomalabnormalities and driver mutations, surface expression of IL-6Rαpredicted the response to metformin therapy (p=−0.81,p=0.0045) (FIG.5A). There was no significant correlation between response to metforminand cell surface expression of IL-6ST (p=−0.14,p=0.7009) (FIG. 5B). Notethat IL-6ST is also known as IL-6R13. To further confirm this, IL-6Rαwas silenced in RPMI cells (FIG. 5C) and the sensitivity of the cells tometformin (IC₅₀) was quantitated. Consistent with the correlation datafrom FIG. 5A, the IC₅₀ for metformin significantly increased in RPMIIL-6Rα knock-downs compared to the scramble control (FIG. 5E). Tofurther confirm this, IL-6Rα was transiently over-expressed in JJN3(FIG. 5D), that naturally expresses very low levels of IL-6-Rα (FIG.4A). Compared to wild type JJN3, the IC₅₀ for metformin significantlydecreased in JJN3 engineered to over-express IL-6Rα (FIG. 5F).

Metformin Killed Myeloma Cells by Down-Regulating IL-6Rα Expression andNegating the Growth and Survival Signal of IL-6 on Myeloma Cells.

To evaluate whether metformin directly modulates IL-6 signaling byaltering the expression of components of the IL-6 signaling cascade,HMCLs were cultured in the presence or absence of metformin and IL-6(ELISA, data not presented), IL-6Rα and IL-6Rβ expression was measuredby flow cytometry. Indeed, metformin significantly and consistentlyreduced cell surface expression of IL-6Rα (FIG. 6A) but not IL-6Rβ (FIG.6B). Concomitantly, no significant change in CD44 expression across thesame HMCLs was found (FIG. 6C). This ruled out global decrease inprotein expression and suggested a direct and specific inhibition ofIL-6Rα expression by metformin. Furthermore, it was investigated whethermetformin induced a decreased in cell surface IL-Rα by inducing sheddingof IL-6Rα as this might decrease classical IL-6 signaling but promotetrans-IL-6 signaling. A decrease in soluble IL-6Rα was observed (FIG.6D) suggesting that the decrease in IL-6Rα expression is at the level ofgene transcription and not due to shedding of IL-6Rα from the cellsurface. To further validate this, IL-6Rα expression was examined at themRNA level after metformin treatment. Metformin decreased the level ofIL-6Rα transcription (FIG. 6E) and therefore, effectively it inhibitsboth classical and trans IL-6 signaling. Additional studies showed thatinhibition of IL-6Rα mRNA levels is due to mIR-34a that is stimulatedvia the down-regulation of the mTOR pathway.

Metformin Kills Primary Myeloma Cells by Down-Regulating IL-6RαExpression.

To validate our observations in HMCLs, in vitro experiments were performon primary myeloma cells isolated from patients with multiple myeloma.The primary myeloma cells studied also covered the known spectrum ofcytogenetic and FISH abnormalities seen with this disease(hyperdiploidy, t(11;14), t(4;14), 17p-, 1q amplification and c-MYCdisruption). The same results were observed as in HMCLs, namely thatmetformin decreased survival/proliferation of primary myeloma cells(FIG. 7A) and decreased cell surface expression of IL-6Rα (FIG. 7B).

Metformin Down-Regulated IL-6Rα Expression on Hepatocytes.

With the discovery of this novel mechanism of action for metformin (i.e.specific down-regulation of IL-6Rα and efficacy against multiplemyeloma), it was determined whether metformin has therapeutic potentialin other disease states where IL-6 may play an important or keypathologic role. Physiologically, IL-6Rα is selectively expressed byB-lymphocytes, plasma cells, hepatocytes and some epithelial cells. Inthe pathogenesis of CRS, IL-6 signaling plays an important role,especially on hepatocytes. The potential of metformin to decrease IL-6Rαexpression on hepatocytes was evaluated. Consistent with theobservations in HMCLs and primary myeloma samples, hepatocytes expressedIL-6Rα and upon metformin treatment, expression of the receptordecreased (FIG. 8A).

Metformin was Effective in Preventing CRP Release from Hepatocytes AfterIL-6 Stimulation.

In patients treated with CAR-T cell therapy or BiTE, CRP levels areroutinely measured in the blood to monitor for CRS. Increased levels ofIL-6 activate IL-6 signaling via IL-6Rα on hepatocytes and in response,CRP is produced by hepatocytes. Since metformin decreased hepatocyteIL-6Rα expression, it was determined whether it could inhibit IL-6signaling and block CRP production. Indeed metformin significantlyinhibited IL-6 induced CRP production from hepatocytes (FIG. 8B).

The Combination of Metformin and Tocilizumab was Superior to EitherAlone in Suppressing CRP Release from the Liver and may be Optimal forPrevention and Therapy of CRS.

At present, tocilizumab is prescribed for the treatment of ongoing CRS.It was examined if metformin will have an additive effect in inhibitingCRP production from hepatocytes in response to IL-6 stimulation. Thecombination of metformin and tocilizumab at the given concentration wereable to reduce CRP levels to baseline suggesting that the combinationcan control CRS significantly better than tocilizumab alone (FIG. 8B).Moreover, pretreatment with metformin may prevent CRS from developing inthe first place.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for treating a mammal having cytokinerelease syndrome (CRS), wherein said method comprises administering tosaid mammal a composition comprising a biguanide, wherein the severityof said CRS is reduced within said mammal.
 2. The method of claim 1,wherein said mammal is a human.
 3. The method of claim 1, wherein saidbiguanide is metformin.
 4. The method of claim 1, wherein said CRS isassociated with an immunotherapy.
 5. The method of claim 4, wherein saidimmunotherapy comprises chimeric antigen receptor T (CAR-T) celltherapy.
 6. The method of claim 4, wherein said immunotherapy comprisesbispecific T-cell engager (BiTE) therapy.
 7. The method of claim 6,wherein said BiTE therapy comprises administering blinatumumab to saidmammal.
 8. A method for treating a mammal at risk of developing CRS,wherein said mammal is scheduled to undergo immunotherapy, wherein saidmethod comprises administering to said mammal a composition comprising abiguanide.
 9. The method of claim 8, wherein said mammal is a human. 10.The method of claim 8, wherein said biguanide is metformin.
 11. Themethod of claim 8, wherein said immunotherapy comprises CAR-T celltherapy.
 12. The method of claim 8, wherein said immunotherapy comprisesBiTE therapy.
 13. The method of claim 12, wherein said BiTE therapycomprises administering blinatumumab to said mammal.
 14. The method ofclaim 8, wherein said composition comprising said biguanide isadministered to said mammal prior to said immunotherapy.
 15. The methodof claim 8, wherein said composition comprising said biguanide isadministered to said mammal concurrent with said immunotherapy.
 16. Themethod of claim 8, wherein said mammal does not develop said CRSfollowing said administering step.
 17. A method for treating a mammalhaving cancer, wherein said cancer comprises cancer cells having IL-6receptor (IL-6R) polypeptides on the surface of said cancer cells,wherein said method comprises administering to said mammal a compositioncomprising a biguanide.
 18. The method of claim 17, wherein said mammalis a human.
 19. The method of claim 17, wherein said IL-6R polypeptidesare IL-6Rα polypeptides.
 20. The method of claim 17, wherein said canceris a blood cancer.
 21. The method of claim 20, wherein said blood canceris a multiple myeloma.
 22. The method of claim 17, wherein said methodfurther comprises administering an agent used to reduce levels of IL-6Rpolypeptides to said mammal.
 23. The method of claim 22, wherein saidagent is an anti-IL-6R antibody.
 24. The method of claim 23, whereinsaid anti-IL-6R antibody is tocilizumab.
 25. The method of claim 17,wherein said mammal is a mammal identified as having said cancer cells.26. The method of claim 17, wherein said method comprises identifyingsaid mammal as having said cancer cells.